A mass-sensitive chemical sensor can be defined as any device that allows for measurement of a property that scales proportionally to mass associated with or bound to a sensing surface of that device. Several such sensor techniques can be utilised, such as evanescent wave-based sensors, e.g. surface plasmon resonance (SPR, which is capable of registering mass changes by the associated change in refractive index at the surface), optical waveguides (also dependent on refractive index changes associated with mass binding events), optical diffraction, optical interference, ellipsometry and acoustic wave devices (for example quartz crystal microbalances (QCMs)). These sensor approaches are well established in the art (see, for example, Biomolecular Sensors, Gizeli and Lowe. Taylor and Francis, London; 2002) and these types of instruments can be used for studies of chemical reactions in situ and for detection of certain molecules in a sample.
A QCM system utilizes the piezoelectric effect of a quartz crystal. In such a system a quartz crystal that is placed between two electrodes, which are connected to an AC-potential, begins to oscillate if the frequency of the AC-potential is close to the resonance frequency of the oscillation mode for the quartz crystal. The resonance frequency of the quartz crystal is a function of many parameters, such as temperature, pressure, cut angle of the crystal, mechanical stress and thickness of the crystal. The resonance frequency is inversely proportional to the thickness of the crystal.
Typical resonance frequencies used in liquid applications range from 1 MHz to 50 MHz. The crystal is normally AT-cut with a circular or square shape with a diameter of approximately 5-10 mm. The electrodes (driving and counter electrodes) are normally of gold on both sides, but other metals are not unusual. The electrodes are very thin compared to the quartz crystal plate and can therefore be considered as part of the crystal plate. When material is added to or removed from one of the electrodes, it becomes thicker or thinner, i.e. the associated weight of the electrode changes. As a consequence of the mass change of the electrode, the resonance frequency of the crystal plate will either decrease or increase and hence the change of resonance frequency can be measured to detect the mass change of the electrode. The mass resolution of a QCM system can be as low as 1 pg/cm2, corresponding to less than 1% of a monolayer of hydrogen.
A typical QCM piezoelectric sensor instrument comprises a sensor element, a sample insertion unit, equipment for determining the piezoelectric properties (including the oscillation frequencies) of a quartz crystal, and signal presentation equipment and buffer and waste containers (other than the sensor element, these items may be referred to as the ‘associated apparatus’ of the sensor instrument). A sample, which can contain any chemical substance of interest, is introduced into the sensor element by the sample insertion unit. The sensor element contains a piezoelectric resonator (the QCM sensor), a sample chamber, flow channels to and from the chamber and an oscillating circuit. The sample induces an interaction with the piezoelectric sensor surface, which can in turn be observed by monitoring the oscillating characteristics of the crystal plate, e.g. by measuring changes in the piezoelectric resonator frequency. The crystal plate is provided with electrical contact areas for the driving and counter electrodes on its surface, such contact areas being connectable to a signal source (e.g. an alternating voltage source) as well as to a measurement device. For measuring, the piezoelectric crystal plate is on one side brought into contact with the fluid (e.g. liquid) sample to be examined. The crystal responds to the accumulation of the mass of the substance to be detected or to a change in the physical properties of the sample by altering its resonance frequency and/or oscillation amplitude.
Piezoelectric sensors can be used for analysis of the viscosity of a liquid sample and are particularly suitable for studying chemical and biochemical interactions. Ha piezoelectric sensor is to be used for the latter purpose, the electrode that is to be exposed to the sample is provided with a specific surface coating, which will interact with the sample. A review of the types of interactions which can be studied using QCM sensors is provided by Cooper and Singleton (J. Mol. Recognit., 2007, 20, 154). Regardless of the type of chemical sensor, common surface coating approaches include self-assembled monolayers (e.g. alkanethiols adsorbed onto gold) and/or polymeric matrices, each of which may bear functional groups which may be used for immobilising a first chemical species of interest. Typically, the immobilised first chemical species is a small organic molecule or an antibody. The sensor bearing the first chemical species is then brought into contact with a dispersion of a second chemical species or a cell and the binding of the second chemical species or cell to the first chemical species is monitored by means of the resultant change in mass at the sensing surface. Fung and Wong (Anal. Chem. 2001, 73, 5302) describe the use of such an approach to detect Salmonella cells in a liquid dispersion, and other similar studies are described by Cooper and Singleton (see above).
A more challenging approach is to employ cells as the immobilised, first species in the chemical sensor. A few studies have achieved this, although the reported methods employ live cells and do not analyse the binding interaction per se; rather these methods use biosensor techniques to monitor post-binding morphological or other changes in the cells (see Marx et al., Anal. Biochem., 2007, 361, 77). Such methods are of little or no use for accurately monitoring the binding interaction, due to interference in the detected signal from the cellular changes following the binding event.
The prior art does not describe or suggest a method for preparing a mass sensitive chemical sensor having immobilised cells and which is suitable for accurately detecting and monitoring a binding interaction between the cells and an analyte ligand.